Tuberculosis (IB) is a persistent lung infection that has plagued mankind for centuries and ranks as one of the most serious threats to world health today. The 2-3 million deaths attributed yearly to the disease, as well as the emergence of strains resistant to all of the available chemotherapeutic agents, urgently call for the development of new therapies to treat TB. Furthermore, the threat of drug-resistant TB as a bioterrorism agent has led to it's listing as a NIAID Category C Priority Pathogen for biodefense research. How the TB bacillus interacts with host cells in order to grow during infection is not well understood. The primary objective of the proposed research is to understand the mechanisms by which this pathogen triggers and manipulates host responses of its primary host cell, the macrophage. From our transcriptional profiling work, we found that M. tuberculosis, a vacuolar pathogen, elicits an response that originates in the cytosol of infected macrophages. Importantly, elicitation of this response depends completely on the bacterial ESX-1 secretion system and is augmented during infections with M. tuberculosis mutants lacking the MmpL4 transporter, providing an unexpected window into microbial players that modulate the host response. We hypothesize that activation of the cytosolic surveillance pathway by M. tuberculosis is important for host control and that genes activated by the pathway work to protect the host from infection. The studies proposed here will test this model and provide molecular details of host-pathogen interactions critical during the early stages of M. tuberculosis infection. Specifically, we will determine the mechanism by which macrophages sense M. tuberculosis infection to initiate the cytosolic surveillance pathway and also identify the bacterial molecule(s) sensed by macrophages and determine how they gain access to the cytosol. Likewise, we will elucidate the regulatory role of the MmpL4 transporter in modulating the host response. Finally, we will integrate experimental and bioinformatic approaches to identify other common, as well as pathogen-specific, responses of murine and human macrophages to infection. Ultimately, by understanding tuberculosis pathogenesis at the molecular level, we hope to aid in the discovery of new therapies to combat and eradicate this persistent infection. Because cytosolic recognition by macrophages is conserved in many bacterial infections, understanding its function may lead to the development of new treatments and diagnostics for a broad range of infectious diseases. Likewise, elucidating the responses specific to individual pathogens may help in the development of pathogen detection schemes and diagnostics.